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Co-localization of Leukotriene A4Hydrolase with 5-Lipoxygenase in Nuclei of Alveolar Macrophages and Rat Basophilic Leukemia Cells but Not Neutrophils

2001; Elsevier BV; Volume: 276; Issue: 37 Linguagem: Inglês

10.1074/jbc.m105676200

1083-351X

Thomas G. Brock, Elana Maydanski, Robert McNish, Marc Peters‐Golden,

Immune Cell Function and Interaction

The synthesis of leukotriene B4 from arachidonic acid requires the sequential action of two enzymes: 5-lipoxygenase and leukotriene A4hydrolase. 5-Lipoxygenase is known to be present in the cytoplasm of some leukocytes and able to accumulate in the nucleoplasm of others. In this study, we asked if leukotriene A4 hydrolase co-localizes with 5-lipoxygenase in different types of leukocytes. Examination of rat basophilic leukemia cells by both immunocytochemistry and immunofluorescence revealed that leukotriene A4 hydrolase, like 5-lipoxygenase, was most abundant in the nucleus, with only minor occurrences in the cytoplasm. The finding of abundant leukotriene A4 hydrolase in the soluble nuclear fraction was substantiated by two different cell fractionation techniques. Leukotriene A4 hydrolase was also found to accumulate together with 5-lipoxygenase in the nucleus of alveolar macrophages. This result was obtained using both in situand ex vivo techniques. In contrast to these results, peripheral blood neutrophils contained both leukotriene A4hydrolase and 5-lipoxygenase exclusively in the cytoplasm. After adherence of neutrophils, 5-lipoxygenase was rapidly imported into the nucleus, whereas leukotriene A4 hydrolase remained cytosolic. Similarly, 5-lipoxygenase was localized in the nucleus of neutrophils recruited into inflamed appendix tissue, whereas leukotriene A4 hydrolase remained cytosolic. These results demonstrate for the first time that leukotriene A4hydrolase can be accumulated in the nucleus, where it co-localizes with 5-lipoxygenase. As with 5-lipoxygenase, the subcellular distribution of leukotriene A4 hydrolase is cell-specific and dynamic, but differences in the mechanisms regulating nuclear import must exist. The degree to which these two enzymes are co-localized may influence their metabolic coupling in the conversion of arachidonic acid to leukotriene B4. The synthesis of leukotriene B4 from arachidonic acid requires the sequential action of two enzymes: 5-lipoxygenase and leukotriene A4hydrolase. 5-Lipoxygenase is known to be present in the cytoplasm of some leukocytes and able to accumulate in the nucleoplasm of others. In this study, we asked if leukotriene A4 hydrolase co-localizes with 5-lipoxygenase in different types of leukocytes. Examination of rat basophilic leukemia cells by both immunocytochemistry and immunofluorescence revealed that leukotriene A4 hydrolase, like 5-lipoxygenase, was most abundant in the nucleus, with only minor occurrences in the cytoplasm. The finding of abundant leukotriene A4 hydrolase in the soluble nuclear fraction was substantiated by two different cell fractionation techniques. Leukotriene A4 hydrolase was also found to accumulate together with 5-lipoxygenase in the nucleus of alveolar macrophages. This result was obtained using both in situand ex vivo techniques. In contrast to these results, peripheral blood neutrophils contained both leukotriene A4hydrolase and 5-lipoxygenase exclusively in the cytoplasm. After adherence of neutrophils, 5-lipoxygenase was rapidly imported into the nucleus, whereas leukotriene A4 hydrolase remained cytosolic. Similarly, 5-lipoxygenase was localized in the nucleus of neutrophils recruited into inflamed appendix tissue, whereas leukotriene A4 hydrolase remained cytosolic. These results demonstrate for the first time that leukotriene A4hydrolase can be accumulated in the nucleus, where it co-localizes with 5-lipoxygenase. As with 5-lipoxygenase, the subcellular distribution of leukotriene A4 hydrolase is cell-specific and dynamic, but differences in the mechanisms regulating nuclear import must exist. The degree to which these two enzymes are co-localized may influence their metabolic coupling in the conversion of arachidonic acid to leukotriene B4. leukotriene 5-lipoxygenase alveolar macrophage diaminobenzidine diamidino-2-phenylindole polymorphonuclear leukocyte rat basophilic leukemia phosphate-buffered saline Leukotriene B4(LTB4)1 is a lipid mediator with important roles in immune defense, inflammation, and disease. For example, LTB4 stimulates chemotaxis (1Ford-Hutchinson A.W. Bray M.A. Doig M.V. Shipley M.E. Smith M.J.H. Nature. 1980; 286: 264-265Crossref PubMed Scopus (1572) Google Scholar), adhesion to endothelium (2Gimbrone M.A.J. Brock A.F. Schafer A.I. J. Clin. Invest. 1984; 74: 1552-1555Crossref PubMed Scopus (152) Google Scholar, 3Tonnesen M. Anderson D. Springer T. Knedler A. Avdi N. Henson P. J. Clin. Invest. 1989; 83: 637-646Crossref PubMed Scopus (181) Google Scholar), degranulation (4Feinmark S.J. Lindgren J.A. Claesson H.E. Malmsten C. Samuelsson B. FEBS Lett. 1981; 136: 141-144Crossref PubMed Scopus (110) Google Scholar, 5Dahlen S.-E. Bjork J. Hedqvist P. Arfors K.-E. Hammarstrom S. Lindgren J.-A. Samuelsson B. Proc. Natl. Acad. Sci. U. S. A. 1981; 78: 3887-3891Crossref PubMed Scopus (788) Google Scholar), superoxide anion generation (6Cunningham F.M. Shipley M.E. Smith M.J. J. Pharm. Pharmacol. 1980; 32: 377-380Crossref PubMed Scopus (46) Google Scholar, 7Claesson H.E. Feinmark S.J. Biochim. Biophys. Acta. 1984; 804: 52-57Crossref PubMed Scopus (30) Google Scholar), and phagocytosis (8Mancuso P. Nana-Sinkam P. Peters-Golden M. Infect. Immun. 2001; 69: 2011-2016Crossref PubMed Scopus (110) Google Scholar) by neutrophils (PMNs). The overproduction of LTB4 plays a role in the pathogenesis of a variety of inflammatory diseases, including glomerulonephritis, rheumatoid arthritis, psoriasis, inflammatory bowel disease, acute lung injury, and interstitial lung disease (9Lewis R.A. Austen K.F. Soberman R.J. N. Engl. J. Med. 1990; 323: 645-655Crossref PubMed Scopus (1162) Google Scholar, 10Henderson W.J. Ann. Intern. Med. 1994; 121: 684-697Crossref PubMed Scopus (581) Google Scholar, 11Goetzl E.J. An S. Smith W.L. FASEB J. 1995; 9: 1051-1058Crossref PubMed Scopus (246) Google Scholar). The first step in the synthesis of LTB4 from arachidonic acid is mediated by the enzyme 5-lipoxygenase (5-LO), which catalyzes the insertion of molecular oxygen into arachidonic acid to form 5-hydroperoxyeicosatetraenoic acid as well as its subsequent dehydration to LTA4 (12Samuelsson B. Funk C.D. J. Biol. Chem. 1989; 264: 19469-19472Abstract Full Text PDF PubMed Google Scholar, 13Needleman P. Turk J. Jakschik B.A. Morrison A.R. Lefkowith J.B. Annu. Rev. Biochem. 1986; 55: 69-102Crossref PubMed Google Scholar). LTA4 is then modified by the epoxide hydrolase activity of the enzyme LTA4 hydrolase to generate LTB4 (14Haeggstrom J.Z. Wetterholm A. Medina J.F. Samuelsson B. J. Lipid Mediat. 1993; 6: 1-13PubMed Google Scholar). Independent of its epoxide hydrolase activity, LTA4hydrolase also has an aminopeptidase activity (15Orning L. Gierse J.K. Fitzpatrick F.A. J. Biol. Chem. 1994; 269: 11269-11273Abstract Full Text PDF PubMed Google Scholar, 16Haeggstrom J.Z. Wetterholm A. Vallee B.L. Samuelsson B. Biochem. Biophys. Res. Commun. 1990; 173: 431-437Crossref PubMed Scopus (112) Google Scholar). Several studies demonstrate that 5-LO is present in the cytoplasm of some cell types and in the nucleoplasm of others (17Brock T.G. Paine R.I. Peters-Golden M. J. Biol. Chem. 1994; 269: 22059-22066Abstract Full Text PDF PubMed Google Scholar, 18Chen X.-S. Naumann T.A. Kurre U. Jenkins N.A. Copeland N.G. Funk C.D. J. Biol. Chem. 1995; 270: 17993-17999Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar, 19Woods J.W. Coffey M.J. Brock T.G. Singer I.I. Peters-Golden M. J. Clin. Invest. 1995; 95: 2035-2040Crossref PubMed Scopus (156) Google Scholar). For example, 5-LO is found in the cytoplasm of peripheral blood neutrophils (17Brock T.G. Paine R.I. Peters-Golden M. J. Biol. Chem. 1994; 269: 22059-22066Abstract Full Text PDF PubMed Google Scholar) but predominantly in the nucleoplasm of alveolar macrophages and rat basophilic leukemia (RBL) cells and mast cells (17Brock T.G. Paine R.I. Peters-Golden M. J. Biol. Chem. 1994; 269: 22059-22066Abstract Full Text PDF PubMed Google Scholar, 18Chen X.-S. Naumann T.A. Kurre U. Jenkins N.A. Copeland N.G. Funk C.D. J. Biol. Chem. 1995; 270: 17993-17999Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar, 19Woods J.W. Coffey M.J. Brock T.G. Singer I.I. Peters-Golden M. J. Clin. Invest. 1995; 95: 2035-2040Crossref PubMed Scopus (156) Google Scholar). Furthermore, 5-LO can be induced to move into the nucleus after various stimuli. For example, 5-LO moves into the nucleus of PMNs after adherence to surfaces or recruitment from the blood into sites of inflammation (20Brock T.G. McNish R.W. Bailie M.B. Peters-Golden M. J. Biol. Chem. 1997; 272: 8276-8280Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar). Similarly, 5-LO moves into the nucleus of eosinophils after adherence (21Brock T.G. Anderson J.A. Fries F.P. Peters-Golden M. Sporn P.H.S. J. Immunol. 1999; 162: 1669-1676PubMed Google Scholar) or in response to treatment with cytokines (22Cowburn A.S. Holgate S.T. Sampson A.P. J. Immunol. 1999; 163: 456-465PubMed Google Scholar, 23Hsieh F.H. Lam B.K. Penrose J.F. Austen K.F. Boyce J.A. J. Exp. Med. 2001; 193: 123-133Crossref PubMed Scopus (177) Google Scholar). None of the above phenomena are associated with enzyme activation. However, after cell stimulation, 5-LO moves from its site in the cytoplasm or nucleoplasm to become reversibly associated with the nuclear envelope and endoplasmic reticulum (24Rouzer C.A. Kargman S. J. Biol. Chem. 1988; 263: 10980-10988Abstract Full Text PDF PubMed Google Scholar, 25Brock T.G. McNish R.W. Peters-Golden M. Biochem. J. 1998; 329: 519-525Crossref PubMed Scopus (33) Google Scholar). The process of membrane association is calcium-dependent (26Rouzer C.A. Samuelsson B. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 7393-7397Crossref PubMed Scopus (94) Google Scholar) and is thought to be essential for the catalytic action of 5-LO. Our current understanding of the enzyme LTA4 hydrolase holds that it is a soluble protein, presumably located within the cytoplasm. Since 5-LO can accumulate in the nucleus of leukocytes, we hypothesized that LTA4 hydrolase might likewise be found within the nucleoplasm. In this study, we demonstrate that the subcellular distribution of LTA4 hydrolase is cell-specific, co-localizing with 5-LO in the nucleoplasm of resting AMs and RBL cells but in the cytoplasm of resting blood PMNs. However, the regulation of nuclear import of LTA4 hydrolase is distinct from that of 5-LO, since only the latter moves into the nucleus after adherence or recruitment of PMNs. F1 male F-344xBN rats at 6 months of age were obtained from the National Institute on Aging. The rats were housed individually in specific pathogen-free conditions for 2 weeks before experimentation. All procedures were performed in accordance with the Guide for the Care and Use of Laboratory Animals as approved by the Council of the American Physiological Society and the University of Michigan Committee on Use and Care of Animals. Rat basophilic leukemia cells (RBL-1, American Type Culture Collection) were seeded at 1 × 10−5 cell ml−1 in minimal essential medium-α (Life Technologies, Inc.) containing 10% fetal calf serum supplemented with penicillin, streptomycin, and amphotericin B (Life Technologies, Inc.). Cells were fed 2 days after seeding and harvested on the third day for experimentation. In some experiments, RBL cells were pelleted and resuspended in medium without serum at 2 × 105 cells ml−1. An equal volume of medium containing 2 μmA23187 was added to stimulate the cells, and the cells were maintained for 5 min at 37 °C. Primary AMs were obtained by lung lavage of 6-month F-344xBN rats by techniques described previously (27Peters-Golden M. McNish R.W. Hyzy R. Shelly C. Toews G.B. J. Immunol. 1990; 144: 263-270PubMed Google Scholar). Human PMNs were isolated from venous blood obtained from healthy volunteers. Purification involved the sequential steps of centrifugation through Ficoll-Paque (Amersham Pharmacia Biotech), dextran sedimentation, and hypotonic lysis of erythrocytes (20Brock T.G. McNish R.W. Bailie M.B. Peters-Golden M. J. Biol. Chem. 1997; 272: 8276-8280Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar). Viability was assessed by trypan blue exclusion. Cells were >95% neutrophils. For adherence, PMNs were placed on fibronectin-coated glass coverslips in Hanks' balanced salts with calcium and magnesium supplemented with 10 mm HEPES for 30 min at 37 °C. Suspension cultured PMNs were maintained in identical conditions in Teflon tubes. The experimental protocol was approved by the University of Michigan Medical School Institutional Review Board for Approval of Research Involving Human Subjects. Appendix tissues were obtained from anonymous human subjects undergoing surgery for purposes unrelated to this study. Tissues were supplied by the Tissue Procurement Core of the University of Michigan Comprehensive Cancer Center. For immunocytochemistry, RBL cells were diluted to 1 × 105 cells ml−1 with serum-free media, mounted on slides by cytospin, and fixed immediately in −20 °C methanol for 30 min. Mounts were then permeabilized in −20 °C acetone for 3 min and air-dried. For immunohistochemistry, formalin-fixed, paraffin-embedded tissue sections were dewaxed in Americlear and rehydrated through decreasing concentrations of ethanol. All materials were then quenched of endogenous peroxidase activity by treatment with 0.3% hydrogen peroxide for 30 min, washed, and blocked with Powerblock (InnoGenex, San Ramon, CA). Primary antibodies, rabbit polyclonal antibodies raised against human 5-LO and LTA4 hydrolase, were a generous gift from Dr. J. Evans, Merck Frosst Center for Therapeutic Research, Pointe Claire-Dorval, Quebec, Canada. Antibodies were prepared in PBS containing 0.1% bovine serum albumin (5-LO 1:750, LTA4 hydrolase 1:1000) and applied overnight at 4 °C. After washing with 0.1% bovine serum albumin in PBS, slides were probed with secondary antibody (biotinylated goat anti-rabbit, 1:250) for 30 min at 37 °C, washed again, then treated with avidin-biotinylated peroxidase complex (Vectastain Elite ABC kit, Vector Laboratories) for 30 min at room temperature. 3,3′-Diaminobenzidine (DAB) was used as peroxidase substrate; some preparations were counterstained with Harris' hematoxylin. Indirect immunofluorescent staining was performed as described previously (28Brock T.G. McNish R.W. Peters-Golden M. J. Biol. Chem. 1995; 270: 21652-21658Abstract Full Text Full Text PDF PubMed Scopus (119) Google Scholar), and after fixation and permeabilization, cells were blocked for 30 min at 37 °C with 0.1% bovine serum albumin in PBS and probed with primary antibodies (5-LO, 1:250; LTA4hydrolase, 1:500; tubulin, 1:1000) at 37 °C for 1 h. Cells were then washed and reprobed with rhodamine-conjugated goat anti-rabbit antibody (titer 1:200), washed, and mounted. In some preparations, nucleic acids were fluorescently labeled using diamidino-2-phenylindole (DAPI). Samples were viewed with a Nikon Eclipse E600 microscope and imaged with a SPOT Slider digital camera using SPOT Advanced software. Confocal microscopy was performed using a Zeiss LSM 510 microscope equipped with LSM software. Enucleation was performed essentially as described (17Brock T.G. Paine R.I. Peters-Golden M. J. Biol. Chem. 1994; 269: 22059-22066Abstract Full Text PDF PubMed Google Scholar). Briefly, PBS-washed RBL cells (1 × 107 cells) were resuspended in 15% Ficoll in minimal essential medium-α containing 20 μg ml−1 cytochalasin B and incubated at 37 °C for 30 min. This was layered atop a discontinuous gradient of 15, 16, 17, 20, and 25% Ficoll in minimal essential medium-α containing 20 μg ml−1 cytochalasin B and 1 μm EGTA and centrifuged at 100,000 ×g for 30 min at 24 °C. Microscopic examination revealed cytoplasts at the 20–25% interface and nucleoplasts in the pellet at the bottom of the Ficoll gradient. Cytoplasts and nucleoplasts were harvested from the gradient, diluted in an equal volume of minimal essential medium-α, centrifuged at 1,000 × g, 10 min, 4 °C, and resuspended in TKM buffer (50 mmTris-HCl, pH 7.4, 25 mm KCl, 5 mmMgCl2) with protease inhibitors (1 mm each phenylmethylsulfonyl fluoride, dithiothreitol, soybean trypsin inhibitor, and leupeptin). An aliquot of the nucleoplasts was sonicated and centrifuged at 100,000 × g for 60 min at 4 °C to obtain nuclear-soluble and nuclear-pelletable fractions. Aliquots of cytoplasts and nucleoplasts were also examined for trypan blue exclusion, counted, and stained for tubulin by indirect immunofluorescence and for DNA with DAPI. Nitrogen cavitation was as described (17Brock T.G. Paine R.I. Peters-Golden M. J. Biol. Chem. 1994; 269: 22059-22066Abstract Full Text PDF PubMed Google Scholar), with PBS-washed cells suspended in ice-cold TKM buffer with protease inhibitors at 107 cells ml−1 and subjected to nitrogen at 350 p.s.i. for 5 min at 4 °C. Total cell breakage was confirmed by the absence of trypan blue exclusion throughout a broad field of cavitate sample. Cavitate was then centrifuged at 1000 ×g for 10 min at 4 °C to pellet nuclei. The low speed supernatant was then centrifuged at 100,000 × g for 60 min at 4 °C to produce cytosolic (supernatant) and non-nuclear membrane (pellet) fractions. The low speed pellet (nuclei) was resuspended in TKM buffer with 0.25 m sucrose, sonicated with 10 brief bursts at a 20% cycle with a Branson sonicator, and centrifuged at 100,000 × g for 60 min at 4 °C to produce nuclear-soluble and -pelletable fractions. In some experiments, 1 mm calcium chloride was included in the TKM buffer throughout the fractionation procedure. A protein assay and immunoblot analysis were performed as described previously (29Peters-Golden M. McNish R. Biochem. Biophys. Res. Commun. 1993; 196: 147-153Crossref PubMed Scopus (176) Google Scholar). Briefly, protein concentration was determined by a modified Coomassie dye binding assay (Pierce), and 10 μg of each sample were separated by SDS-polyacrylamide gel electrophoresis, transferred to nitrocellulose, blocked with 5% nonfat dry milk, probed with primary antibody (5-LO, 1:3000; LTA4H, 1:5000), washed, and reprobed with secondary antibody (horseradish peroxidase-linked goat anti-rabbit antibody, 1:5,000). Detection was by ECL chemiluminescent reagent (Amersham Pharmacia Biotech) and Hyperfilm chemiluminescent film (Amersham Pharmacia Biotech). The subcellular distribution of LTA4 hydrolase as well as 5-LO was determined first in the RBL-1 cell line, which can generate significant amounts of LTB4 (30Ford-Hutchinson A.W. Piper P.J. Samhoun M.N. Br. J. Pharmacol. 1982; 76: 215-220Crossref PubMed Scopus (20) Google Scholar). By immunocytochemistry, positive staining for LTA4 hydrolase was evident in both the cytoplasm and nucleus, with the nucleus staining more intensely than the cytoplasm (Fig.1). Essentially identical results were obtained for 5-LO; weak positive staining was present in the cytoplasm, whereas the nucleus showed much stronger staining. Cells probed in parallel with non-immune serum did not demonstrate any staining. Indirect immunofluorescent microscopy was used as a second approach to localize LTA4 hydrolase and 5-LO in individual cells; it avoids potential artifacts due to endogenous peroxidase activity, as may occur with immunocytochemistry, and it allows dual staining for DNA. By this method, the cytoplasm stained poorly for LTA4hydrolase and 5-LO, whereas the nucleus stained strongly for both (Fig.1). Nuclear staining with DAPI supported localization of both LTA4 hydrolase and 5-LO to the nucleus. Curiously, the overlay of rhodamine and DAPI signals showed both overlapping and distinct signals for both enzymes, suggesting that the distribution of either enzyme was identical neither to the DNA distribution nor to that of the other enzyme. Although 5-LO has been shown to be soluble within the nucleoplasm of resting cells (17Brock T.G. Paine R.I. Peters-Golden M. J. Biol. Chem. 1994; 269: 22059-22066Abstract Full Text PDF PubMed Google Scholar, 20Brock T.G. McNish R.W. Bailie M.B. Peters-Golden M. J. Biol. Chem. 1997; 272: 8276-8280Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar), it was not clear from the above analysis whether LTA4 hydrolase was also within the nucleoplasm of RBL cells or simply associated with the nuclear envelope. To address this question, RBL cells dual-stained for LTA4 hydrolase and DNA were imaged by confocal microscopy. When serial optical sections for both stains were collected simultaneously, LTA4 hydrolase was found to co-localize with DNA within the nucleus (Fig. 2). However, as described above, the two signals were not absolutely identical, indicating that the distribution of LTA4 hydrolase did not completely match that of the DNA. In separate experiments involving confocal imaging of 5-LO and DNA distribution in RBL cells, 5-LO was also found to be intranuclear but not identical in distribution to that of DNA (data not shown). Although the above techniques indicate immunodetectable protein within nuclei, they cannot determine if the nuclear protein is soluble and the same molecular weight as the cytosolic protein. As one approach to assess these issues, RBL cells were treated with cytochalasin b and centrifuged through a discontinuous Ficoll gradient. By this technique, >90% nucleus-free cytoplasts were obtained at the 20–25% Ficoll interface, as indicated by positive staining for the cytosolic marker α-tubulin (Fig. 3 B), negative staining for DAPI (not shown), and positive trypan blue exclusion (not shown). Nucleoplasts, characterized as α-tubulin negative (Fig. 3 B), DAPI positive (not shown), and trypan blue staining (not shown) were obtained in the pellet. By immunoblot analysis, both cytoplasts and nucleoplasts contained the 78-kDa 5-LO protein and the 69-kDa LTA4 hydrolase protein (Fig.3 A). As shown previously (17Brock T.G. Paine R.I. Peters-Golden M. J. Biol. Chem. 1994; 269: 22059-22066Abstract Full Text PDF PubMed Google Scholar), nucleoplasts from RBL cells contain 5-LO in both soluble and pelletable fractions. In contrast, LTA4 hydrolase is restricted to the soluble phase of RBL nucleoplasts (Fig. 3 A). We have previously demonstrated that, under proper conditions, nitrogen cavitation can effectively disrupt cells while maintaining nuclear integrity (17Brock T.G. Paine R.I. Peters-Golden M. J. Biol. Chem. 1994; 269: 22059-22066Abstract Full Text PDF PubMed Google Scholar). When this is done in the absence of calcium, 5-LO protein is most abundant in the cytosolic- and nuclear-soluble fractions, although it is also detectable in membrane and nuclear pelletable fractions (Fig. 4 Aand Refs. 17Brock T.G. Paine R.I. Peters-Golden M. J. Biol. Chem. 1994; 269: 22059-22066Abstract Full Text PDF PubMed Google Scholar and 28Brock T.G. McNish R.W. Peters-Golden M. J. Biol. Chem. 1995; 270: 21652-21658Abstract Full Text Full Text PDF PubMed Scopus (119) Google Scholar). By this technique (and in the same fractions), LTA4 hydrolase is also predominantly in the cytosolic- and nuclear-soluble fractions, although minor amounts can also be detected in the membrane fraction (Fig. 4A). Because 5-LO can undergo calcium-dependent membrane association after cell stimulation (24Rouzer C.A. Kargman S. J. Biol. Chem. 1988; 263: 10980-10988Abstract Full Text PDF PubMed Google Scholar, 31Kargman S. Prasit P. Evans J.F. J. Biol. Chem. 1991; 266: 23745-23752Abstract Full Text PDF PubMed Google Scholar) and because 5-LO and LTA4 hydrolase function sequentially in LTB4synthesis, it was of interest to determine if LTA4hydrolase could demonstrate either calcium-dependent membrane association or membrane association after cell stimulation. As expected, when cells were fractionated in the presence of calcium, the majority of 5-LO protein was found to be associated with the membrane and nuclear-pelletable fractions (Fig. 4 A). However, in the same samples, LTA4 hydrolase remained predominantly soluble, with a distribution among fractions which matched that found in cells not given calcium. When intact RBL cells were stimulated with 1 μmA23187 for 5 min, immunofluorescent detection showed that 5-LO appeared to translocate to nuclear membranes, whereas no change in intranuclear distribution of LTA4 hydrolase was found (Fig. 4 B). To determine if nuclear co-localization of LTA4hydrolase with 5-LO extended to primary leukocytes, we examined rat AMs, which like RBL cells, can synthesize abundant LTB4(27Peters-Golden M. McNish R.W. Hyzy R. Shelly C. Toews G.B. J. Immunol. 1990; 144: 263-270PubMed Google Scholar). Lung tissue was harvested from normal, untreated rats, inflated and fixed overnight with formalin. Tissue slices were then stained to compare the subcellular localization of LTA4 hydrolase with that of 5-LO in AMs in situ. Positive staining for LTA4 hydrolase, indicated by the brown color developed from the peroxidase substrate DAB, was apparent in both the cytoplasm and the nucleus of AMs, with the nucleus staining being consistently darker (Fig. 5, large arrowhead). This contrasts with the staining of alveolar epithelial cells (Fig. 5,small arrowheads), in which brown staining for hydrolase was present in the cytoplasm but absent from the nuclei, which were light blue due to counterstaining with hematoxylin. Staining for 5-LO in AMs within a serial section was, like that for hydrolase, heaviest within the nucleus but also evident within the cytoplasm. In serial sections probed with non-immune serum, brown staining was negligible. The subcellular distribution of LTA4 hydrolase was also assessed in AMs isolated from the lung by lavage. By indirect immunofluorescent microscopy, LTA4 hydrolase, like 5-LO, was accumulated within the nucleus, although light staining for both enzymes was also detectable within the cytoplasm (Fig.6). Nuclear localization within given fields was confirmed by positive staining with DAPI. Previously, we have shown that 5-LO is found predominantly in the cytosolic- and nuclear-soluble fractions of AMs broken by nitrogen cavitation (28Brock T.G. McNish R.W. Peters-Golden M. J. Biol. Chem. 1995; 270: 21652-21658Abstract Full Text Full Text PDF PubMed Scopus (119) Google Scholar). Similarly, LTA4 hydrolase was most abundant in the same fractions of rat AMs broken and fractionated in the same way (Fig.7).Figure 7Distribution of LTA4 hydrolase in rat AM subcellular fractions. Freshly isolated AMs were subjected to nitrogen cavitation, with subsequent fractionation by differential centrifugation as described under "Experimental Procedures." Fractions were then probed for LTA4 hydrolase by immunoblot analysis. Fractions are cytosolic (C), non-nuclear membrane (M), nuclear soluble (Ns), and nuclear pelletable (Np).View Large Image Figure ViewerDownload Hi-res image Download (PPT) In addition to RBL cells and AMs, PMNs synthesize abundant LTB4. However, unlike RBL cells and AMs, PMNs from peripheral blood have 5-LO solely in the cytoplasm, and the import of 5-LO into the nucleus occurs after adherence of PMNs to surfaces or recruitment of PMNs into sites of inflammation (20Brock T.G. McNish R.W. Bailie M.B. Peters-Golden M. J. Biol. Chem. 1997; 272: 8276-8280Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar). When peripheral blood PMNs were maintained in suspension and probed for LTA4 hydrolase as well as 5-LO protein by indirect immunofluorescent microscopy, both proteins were found to be exclusively cytosolic (Fig.8). When PMNs were allowed to adhere to a fibronectin-coated surface for 30 min, 5-LO accumulated strongly within the multi-lobed nuclei, whereas LTA4 hydrolase remained outside the nucleus (Fig. 8). The subcellular distributions of LTA4 hydrolase and 5-LO in PMNs were also determined in situ using immunohistochemistry applied to tissue sections. In PMNs situated in vessels in healthy rat lung, positive staining for 5-LO (Fig.9 A) and LTA4hydrolase (Fig. 9 B) was restricted to the cytoplasm. When sections of tissue from inflamed appendix were probed for 5-LO, strong nuclear staining of recruited polymorphonuclear cells was evident (Fig.9 C), as expected. Serial sections from the same inflamed tissue probed for LTA4 hydrolase showed the darkest staining associated with polymorphonuclear cells, with lighter positive staining associated with mesenchymal tissue and epithelial cells (Fig.9, D and E). In recruited polymorphonuclear cells as well as other tissue cells, hydrolase staining was predominantly cytosolic. Thus, LTA4 hydrolase can co-localize with 5-LO in the cytoplasm, as in peripheral blood PMNs, or it can segregate from 5-LO, as when it is cytosolic and 5-LO is intranuclear in recruited PMNs. In the present study, we have used multiple techniques to compare the subcellular distribution of two enzymes, 5-LO and LTA4hydrolase, which work sequentially to synthesize LTB4 from arachidonic acid. We have shown for the first time that LTA4 hydrolase can be found in the nucleus, where it co-localizes with 5-LO in resting AMs and RBL cells. In both cell types, lesser amounts of LTA4 hydrolase and 5-LO were also present in the cytoplasm. We also have found that LTA4hydrolase co-localizes with 5-LO in the cytoplasm of peripheral blood PMNs. However, in this cell type, LTA4 hydrolase does not move into the nucleus with 5-LO after adherence or recruitment of these cells. Finally, we have demonstrated that, unlike 5-LO, LTA4 hydrolase does not show calcium-dependent membrane association or translocation after cell stimulation. The finding that LTA4 hydrolase can accumulate in the nucleus of AMs and RBL cells is particularly surprising, since the current thought is that this enzyme is exclusively cytoplasmic. Indeed, the current characterization for this protein in the Swiss-Prot data bank gives its subcellular distribution as "cytoplasmic." Consistent with this characterization, epithelial and mesenchymal cells in Figs. 5 and 9 lack nuclear staining for LTA4 hydrolase. This suggests that only certain cell types may show nuclear import of LTA4 hydrolase. Nuclear import of small molecules may proceed by diffusion, whereas nuclear import of larger molecules, like the 69-kDa LTA4hydrolase, requires the presence of a nuclear import sequence, which typically consists of a cluster of basic amino acids (32Gorlich D. Kutay U. Annu. Rev. Cell Dev. Biol. 1999; 15: 607-660Crossref PubMed Scopus (1651) Google Scholar). Not surprisingly, LTA4 hydrolase has several such clusters, most notably at (human) 186RKIYK. Interestingly, one of the structural domains of LTA4 hydrolase, noted by Haeggstrom and co-workers (33Thunnissen M.M. Nordlund P. Haeggstrom J.Z. Nat. Struct. Biol. 2001; 8: 131-135Crossref PubMed Scopus (254) Google Scholar), features armadillo repeats. These structural elements are also found on the nuclear import-mediating importin proteins, suggesting the possibility that LTA4 hydrolase could instead directly interact with importin-β or the nuclear pore complex. Activation of a nuclear import sequence typically involves a phosphorylation step (34Jans D.A. Hubner S. Physiol. Rev. 1996; 76: 651-685Crossref PubMed Scopus (383) Google Scholar) as a second requirement for nuclear import. Although there is evidence that LTA4 hydrolase can be phosphorylated at serine 415 (35Rybina I.V. Liu H. Gor Y. Feinmark S.J. J. Biol. Chem. 1997; 272: 31865-31871Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar), it is unclear whether this specific phosphorylation event has any effect on subcellular distribution. Clearly, much work is necessary to better understand the mechanism of nuclear import of LTA4 hydrolase. The regulation of the subcellular distribution of LTA4hydrolase can be important in many ways. Most obviously, it may affect LTB4 synthesis. Both AMs and RBL cells, which co-localize 5-LO with LTA4 hydrolase, efficiently metabolize LTA4 to LTB4, as indicated by their ability to secrete large amounts of LTB4 with negligible amounts of LTA4. In contrast, PMNs, which can readily segregate 5-LO from LTA4 hydrolase, secrete significant amounts of LTA4 as well as LTB4 (36Sala A. Bolla M. Zarini S. Muller-Peddinghaus R. Folco G. J. Biol. Chem. 1996; 271: 17944-17948Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar). It is possible that part of the explanation for the inefficient conversion of LTA4 to LTB4 in PMNs lies in the spatial separation of 5-LO and LTA4 hydrolase. Another intriguing role for nuclear import of LTA4hydrolase may relate to regulating the levels of LTA4within the nucleus. Since 5-LO can accumulate in the nucleus, significant amounts of its end product, LTA4, will be generated within that compartment. LTA4 apparently has a striking capacity to bind to constituents of DNA (37Reiber D.C. Murphy R.C. Arch. Biochem. Biophys. 2000; 379: 119-126Crossref PubMed Scopus (17) Google Scholar). This in turn may have the potential to affect transcriptional events. By facilitating the conversion of LTA4 to LTB4, the co-localization of LTA4 hydrolase with 5-LO in the nucleus might reduce the amount of intranuclear LTA4 available to bind to DNA. Finally, nuclear import of LTA4 hydrolase might be relevant to the aminopeptidase function of this enzyme. Although mice that were deficient for LTA4 hydrolase revealed no evidence for an aminopeptidase role for the enzyme (38Byrum R.S. Goulet J.L. Snouwaert J.N. Griffiths R.J. Koller B.H. J. Immunol. 1999; 163: 6810-6819PubMed Google Scholar), it is possible that such a function only becomes apparent when the enzyme is in a particular subcellular locale. This possibility will require additional investigation. In summary, we have shown that LTA4 hydrolase can reside, with 5-LO, within the nucleoplasm of AMs and RBL cells. Moreover, the import of LTA4 hydrolase into the nucleus does not necessarily coincide with that of 5-LO, as can be seen in adherent and recruited PMNs. These results indicate that the nuclear import of LTA4 hydrolase is a regulated event that only occurs in some cell types under specific conditions. LTA4 hydrolase is ubiquitously expressed in non-leukocytic cell types as well, and its import in such cell types remains to be evaluated. Furthermore, failure to correctly regulate the subcellular distribution of LTA4hydrolase may play a role in disease. These possibilities will be the subject of future investigations.